Oxazolidinone derivatives: Cytoxazone-Linezolid hybrids induces apoptosis and senescence in DU145 prostate cancer cells

Article abstract of DOI:10.1016/j.ejmech.2014.04.062

In this study, we report the synthesis of novel oxazolidinone derivatives derived from linezolid 3 having p-methoxyphenyl group at C-4 position. In vitro evaluation for their anticancer activity toward cultured A549, DU145, HELA, and MCF7 were carried out

Full text of DOI:10.1016/j.ejmech.2014.04.062

Accepted Manuscript
Oxazolidinone derivatives: Cytoxazone-Linezolid hybrids induces apoptosis and
senescence in DU145 prostate cancer cells
Annavareddi Naresh, Maddimsetti Venkateswara Rao, Sudha Sravanti Kotapalli,
Ramesh Ummanni, Batchu Venkateswara Rao
PII:
S0223-5234(14)00383-3
10.1016/j.ejmech.2014.04.062
EJMECH 6933
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 January 2014
Revised Date: 20 March 2014
Accepted Date: 22 April 2014
Please cite this article as: A. Naresh, M. Venkateswara Rao, S.S. Kotapalli, R. Ummanni, B.
Venkateswara Rao, Oxazolidinone derivatives: Cytoxazone-Linezolid hybrids induces apoptosis and
senescence in DU145 prostate cancer cells, European Journal of Medicinal Chemistry (2014), doi:
10.1016/j.ejmech.2014.04.062.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Oxazolidinone derivatives: Cytoxazone-Linezolid hybrids induces
apoptosis and senescence in DU145 prostate cancer cells
Annavareddi Naresha, Maddimsetti Venkateswara Raoa, Sudha Sravanti Kotapallib, Ramesh
Ummannib*, Batchu Venkateswara Raoa*
A novel series of oxazolidinones having p-methoxy phenyl group at C-4 position have been
synthesized. Compound 17 induces cellular senescence and apoptosis in DU145 prostate
cancer cells
.
O
O
N
O
O
N
N
O
N
O
F
F
R2
R1
I
O
II
O
13 R2 = CH=CH2, 14 R2 = CH2OH
15 R2 = CH2OCOC6H5
16 R2 = CH2OCO(CH2)2OCOCH=CH2
17 R2 = CH2CH3, 18 R2 = CH2CH2OH
20 R2 = CH2NH2, 21 R2 = CH2NHCOCH3
9 R1 = CH2OH
10 R1 = CH2OCO(CH2)2OCOCH=CH2
11 R1 = CH2OCOC6H5
Cellular senescence
________________________________________________________________________________________________________________

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•
•
•
•
•
A novel series of oxazolidinones have been synthesized.
The series of compounds were evaluated for their anticancer potential.
Couple of compounds exhibited significant inhibitory activity against cancer cells.
Compound 17 induces cellular senescence and apoptosis of DU145 cells.
This study finds oxazolidinones as novel anticancer agents.

ACCEPTED MANUSCRIPT
Oxazolidinone derivatives: Cytoxazone-Linezolid hybrids induces
apoptosis and senescence in DU145 prostate cancer cells
Annavareddi Naresha, Maddimsetti Venkateswara Raoa, Sudha Sravanti Kotapallib, Ramesh
Ummannib*, Batchu Venkateswara Raoa*
aOrganic and Biomolecular Chemistry Division, CSIR-Indian Institute of Chemical
Technology, Hyderabad 500007, India
bCentre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad
500007, India
Abstract: In this study, we report the synthesis of novel oxazolidinone derivatives derived
from linezolid 3 having p-methoxyphenyl group at C-4 position. Invitro evaluation for their
anticancer activity toward cultured A549, DU145, HELA, and MCF7 were carried out. The
series of compounds prepared displayed wide range of cytotoxicity in MTT assays (10 to 70
ꢀM) across the cell lines tested. Of the all tested compounds 16 and 17 displayed good
anticancer potential against A549 (lung) and DU145 (prostate) cancer cells. Further, to
determine their anticancer potential, in the present study we have assessed effect of 17 on
DU145 cells growth in in vitro assays. The results clearly demonstrated that the exposure of
DU145 cells to 17 inhibits cell proliferation and induces apoptosis by activation of caspase -3
and -9. Long term exposure of DU145 cells to 17 induced cellular senescence confirmed by
senescence marker β-galactosidase staining of cells on post exposure to 17. The results from
this current report support that the oxazolidinone derivatives with ethyl and acryl
substitutions showed promising anticancer activity which will be helpful to develop further
novel anticancer agents with better therapeutic potential.
Key words: oxazolidinones, cytoxazone, linezolid, anticancer, apoptosis, senescence.
*aCorresponding author: e-mail:venky@iict.res.in, drb.venky@gmail.com
Tel: +91-40-27193003; Fax: +91-40-27193003.
*bCorresponding author: e-mail:ummanni@iict.res.in
Tel: +91-40-27191866; Fax: +91-40-27193003.
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1. Introduction:
The leading cause of death in the world is cancer. Over 8.0 million people died of
cancer and many more going to get effected with this disease in future [1]. The preliminary
cancer treatment options remains either alone or in combination of radio therapy, surgery and
chemotherapy. For the last several years continuing efforts are being made by industry and
academia to develop useful chemotherapeutic agents. In this connection many class of
compounds have been identified for their antitumor activity.
Oxazolidinone derivatives have a novel mechanism of action on cancer, HIV, monoamine
oxidise, glutamate receptor antagonist, and metabotropic [2]. These are also new class of
antibacterial agents and demonstrate potent in vitro and in vivo activity against important
human pathogens, including multiple antibiotic-resistant strains of gram positive organisms
[3]. Design of new oxazolidinones with different structural modifications for better
therapeutic activity is a very active ongoing research [4]. Some of the oxazolidinones like “3-
nitro-5–methyl-2-oxazolidone,
3-(2-hydroxy-3-(2-nitro-1H-imidazol-1-yl)propyl)-2-
oxazolidinone, (4-(4-(bis(2-chloroethyl)amino)-2,5-dimethoxyphenyl)methylene
aminophenyl)-2-oxazolidinone” are showing anticancer activity and are in early clinical trials
[5]. Posizolid 1, torezolid 2, linezolid 3, cytoxazone 4 and epi-cytoxazone 5 are showing
interesting biological activities having the basic common oxazolidinone structure (Figure 1).
Posizolid 1 is an antibiotic under investigation by AstraZeneca for the treatment of bacterial
infections [6]. Torezolid 2 has been used for complicated skin infections [7]. Linezolid 3 is
the first example of new class of synthetic antibacterial compounds which block protein
biosynthesis and used for the treatment of serious infections caused by Gram-positive
bacteria [8]. Until now a number of structural modifications of linezolid 3 have been
reported. Many research groups involved in the introduction of heterocyclic moieties
replacing the rings A-, B-, or C [9] (Figure 2). Cytoxazone 4 is a natural product shows
cytokine-modulating activity by inhibiting the signalling pathway of Th2 cells [10] and its C-
5-epimer epi-cytoxazone 5, which is a synthetic derivative, is also showing interesting
activity [11]. One of the basic structural differences between cytoxazone 4 and other above
compounds is the presence of p-methoxyphenyl group at C-4 position in the oxazolidinone
ring. We envisaged that the hybrid structures I & II of linezolid 3 & cytoxazone 4 and
linezolid 3 & epi-cytoxazone 5 may be of some importance for new leads, since such kind of
oxazolidinones to the best of our knowledge have not been synthesized. In addition a
substituent at C-4 position creates a new stereo centre which gives the possibility of erythro
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and threo oxazolidinones and helps in studying the relation of stereochemistry with
therapeutic action. In this context here in we describe the synthesis and anticancer activity of
new hybrids having the p-methoxyphenyl functionality at the C-4 position.
2. Results and discussion
2.1. Chemistry
The retrosynthetic analysis for our new hybrid structures I (9, 10, 11) and II (13, 14, 15, 16,
17, 18, 20, 21) were shown in Scheme 1. The required starting materials for our synthesis are
cytoxazone 4 and epi-cytoxazone precursor 12 which have been synthesized using our earlier
procedure [12]. The basic core structures of erythro I and threo II can be prepared by N-
arylation of oxazolidinones 4 & 12 with iodo derivative 6 to give 9 & 13 and further
manipulations of 9 & 13 should give the compounds 10 to 21.
The desired iodo compound 6 (Scheme 2) for the synthesis of main scaffold for Buchwald
coupling can be synthesized from 7 which in turn can be prepared from commercially
available 1,2-difluoro-4-nitrobenzene [13]. The amine in compound 7 was converted to iodo
by diazotization followed by iodination [14] to afford the compound 7 in 85 % yield.
Treatment of cytoxazone 4 with TBSCl afforded the compound 8 in 94 % yield. The coupling
between oxazolidinone 8 (Scheme 3) and iodo compound 6 in Buchwald conditions followed
by deprotection of TBS-group with TBAF solution furnished the compound 9 in 80 % yields
[15]. The compound 9 was treated with benzoic anhydride to give the compound 11 in 90 %
yield. When 9 was treated with acrylic acid, DCC and DMAP interestingly it gave the
compound 10 exclusively in 87 % [16].
For preparing threo derivatives, the coupling (Scheme 4) between oxazolidinone 12 and iodo
compound 6 have been achieved by Buchwald conditions to yield the scaffold 13 in 90%
yield. The olefin in compound 13 was oxidised by O3 in dichloromethane at -78 ºC and then
reduced by the NaBH4 in methanol to afford the compound 14 in 87% yield. The compound
14 was made bezoyl derivative 15 (Scheme 5) by treating with benzoic anhydride and
triethylamine in 77% yield. The compound 14 was made acryloyl derivative using acrylic
acid, DCC and DMAP to give the compound 16 in 85%. The reduction of olefin in compound
13 (Scheme 6) with H2 and Pd/C in MeOH gave the compound 17 in 85% yield and oxidation
of compound 13 with BH3.DMS gave the compound 18 in 75% yield
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The alcohol functionality in compound 14 (Scheme 7) was converted into amine by following
transformations. The hydroxyl in 14 was initially converted into mesyl derivative by
treatment with methanesulfonyl chloride and triethylamine. The mesyl compound was then
treated with NaN3 in DMF at 100 °C to afford the azide 19 in 86% yield. Then the reduction
of azide with H2, Pd/C in methanol gave the amine 20 in 73% yield. The amine in compound
20 was converted into acetyl derivative by treating with acetic anhydride and triethylamine to
afford the compound 21 in 85%.
2.2. Biological study
The series of compounds synthesized were subjected to random screening against selected
cell lines to determine their anticancer potential. The IC50 values of epimers 4 & 5, 9 & 14,
10 & 16 and 11 & 15 have been compared, but significant difference is observed only in 10
& 16. Of all the compounds analyzed, 16 and 17 displayed more cytotoxic effect against
DU145 with an IC50 value of 8.69±0.38 and 9.47±0.18 µM respectively (Table 1). Thus, the
effect of this compound on prostate cancer cell line DU145 was further tested to study its
mechanism of action. As the 17 compound was found to be cytotoxic against DU145 cell
line, the change in morphology induced by this compound was also observed. The inherent
morphology of cells was changed and eventually detached from surface upon exposure at
higher concentration of the compound (Figure 3). Further to access the long term effect of 17
on the anchorage-independent proliferative capacity of the prostate cancer cell line DU145, a
colony formation assay (soft agar assay) mimicking in vivo environment was performed [17].
The results of the assay clearly indicate that the compound 17 has an inhibitory effect on
colony formation ability of proliferating cancer cells (Figure 4). A dose dependent inhibition
of colony formation ability of cells suggests that the compound 17 effect anchorage
independent growth of DU145 cells. In order to confirm whether 17 cause any cell death of
DU145 cells, cell death and cell cycle analysis was performed using a Flow Cytometer. Upon
treatment with 17, DU145 cells accumulated in G0/G1 phase of cell cycle (Figure 5). The
results showing growth arrest in sub G1 indicates cell death upon exposure to 17. The
observed cell death may be due to the apoptosis induced by activation of caspases (Table 2).
Therefore to determine whether the observed cell death is due to activation of caspases which
are prerequisite for the induction of apoptosis eventually leading to cell death. Therefore, we
have measured activation of key caspases by 17. The results have confirmed the activation
both caspase-3 and caspase-9 in DU145 cells treated with 17 for 48 h continuously. The
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activation of procaspase-3 is a distinguishing feature of apoptotic cells. Thus, the presence of
activated caspase-3 was experimentally shown using a fluorogenic substrate Ac-DEVD-
AMC. A dose-dependent increase in the activity of caspase was observed with different
concentrations of 17 as compared to those DU145 cells which were treated with DMSO
(Figure 6A). Similarly, activation of caspase-9 was also shown using a fluorogenic substrate
Ac-DEVD-AFC. Activation of caspase-9 is mitochondrial dependant which in turn activates
caspase-3 executing apoptotic cell death in cancer (Figure 6B). Doxorubicin was used as
positive control for measurement of caspase activation. Taken together the results clearly
show that the compound 17 induced cell death thorough mitochondrial intrinsic pathway. To
further ascertain this claim, the effect of compound 17 on the mitochondrial membrane
potential was observed using a dye called JC-1. The unique property of this dye helps to
determine the changes in the mitochondrial membrane potential. Healthy cells are known to
show abundant red fluorescence (Rhodamine) due to the localization of JC-1 in the inner
mitochondrial membrane matrix. Upon treatment with compound 17, a change in the
mitochondrial membrane potential was observed as a result of the dye released from the
mitochondrial matrix into the cytosol leading to a shift from red to green fluorescence (FITC)
(Figure 7). The loss of mitochondrial membrane potential is known to release cytochrome c
in turn activating caspase-9. In summary these observations suggest that the exposure of
DU145 cells to 17 leads to mitochondrial dependent induction of apoptosis eventually leading
to cell death.
Though we observed little extent of apoptosis in DU145 cells exposed to 17, proliferation
assays show strong effect on total growth of cells. Certain small molecules possess the ability
to induce senescence in healthy cells. To test whether 17 had a similar effect on prostate
cancer cell line DU145, senescence SA-βgal assay was performed. This is a cytochemical
assay wherein the chromogenic substrate 5-bromo-4-chloro-3-indoyl β-D-galactopyranoside
(X-gal) yields an insoluble blue compound when cleaved by β-galactosidase increased in
senescent cell. Interestingly, this activity is not observed in healthy or quiescent cells, thus,
making SA-βgal a biomarker for senescent cells. In the present study as observed in figure 7
up on treatment with 17 increased SA-βgal staining in DU145 cells strongly suggest that 17
induces senescence of DU145 cells there by inhibiting their growth (Figure 8).
3. Conclusion
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In conclusion, a series of novel oxazolidinone derivatives has been synthesised and
characterized. Evaluation for their anticancer activity toward cultured A549, DU145, HELA,
and MCF7 were carried out. From the series of compounds synthesized in the present study,
compound 17 affecting prostate cancer cell proliferation partly by direct targeting cell cycle
progression and inducing cellular senescence. This from all above experiments it is evident
that the cytoxazone-linezolid hybrids with acryl or ethyl substitutions showed more
promising anticancer potential than the other substitutions reported. We hypothesize that the
compound 17 may target many pro-survival pathways in cancer cells shows aptness to
consider this scaffold as basis to develop novel an anticancer agent.
4. Experimental protocols
4.1. General
TLC was performed on Merck Kiesel gel 60; F254 plates (layer thickness 0.25 mm). Column
chromatography was performed on silica gel (60–120 mesh) using ethyl acetate and hexane
mixture as eluent. Melting points were determined on a Fisher John’s melting point
1
apparatus. IR spectra were recorded on a Perkin–Elmer RX-1 FT-IR system. H NMR and
13C NMR spectra were recorded using Bruker Avance-300 MHz, Varian-400, and 500 MHz
spectrometers. 1H NMR data are expressed as chemical shifts in ppm followed by multiplicity
(s-singlet; d-doublet; t-triplet; q-quartet; m-multiplet), number of proton (s) and coupling
constant (s) J (Hz). 13C NMR chemical shifts are expressed in ppm. Optical rotations were
measured with JASCO digital polarimeter. Accurate mass measurement was performed on Q
STAR mass spectrometer (Applied Biosystems, USA).
4.2. Synthesis of 4-(2-fluoro-4-iodophenyl)morpholine (6):
To a solution of p-TsOH·H2O (11.63 g, 61.22 mmol) in acetonitlile (40 mL) was added the
compound 7 (4 g, 20.4 mmol). The resulting suspension was cooled to 10–15 °C and to this
was added, gradually, a solution of NaNO2 (2.81 g, 40.81 mmol) and KI (8.46 g, 51 mmol) in
water (10 mL). The reaction mixture was stirred for 10 minutes then allowed to come to 20
°C and stirred until the starting material consumed. To the reaction mixture was then added
water (100 mL), NaHCO3 (1 M; until pH = 9–10) and Na2S2O3 (2M, 40 mL). The aqueous
layer was extracted with ethyl acetate (2
X 50 mL) and the combined organic layers were
washed with water and brine, dried over anhydrous Na2SO4. The solvent was removed on a
rotary evaporator and the residue was purified by column chromatography (ethyl
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acetate/hexane, 1:4) to give compound 6 (5.325 g, 85%) as white solid. mp: 85–87 °C, FT-IR
(neat) vmax cm-1 2960, 2853, 1558, 1490, 1372, 1334, 1296, 1255, 1234, 1165, 1112, 922,
1
856, 805, 751, 573. H NMR (CDCl3, 500 MHz) : δ 7.39-7.33 (m, 2H), 6.66 (t, 1H, J = 8.6
Hz), 3.87-3.84 (m, 4H), 3.07-3.04 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ 155.2 (d, J = 251.3
Hz), 139.9 (d, J = 8.2 Hz), 133.5 (d, J = 3.8 Hz), 125.1 (d, J = 23.6 Hz), 122.6 (d, J = 7.6
Hz), 120.2 (d, J = 2.7 Hz), 66.8, 50.59, 50.55. ESIMS m/z: 308 [M+H] +, HRMS (ESI) Calcd
for C10H12ONFI [M+H]+ 307.99272, found 307.99321.
4.3.
Synthesis
of
(4R,5R)-5-(((tert-butyldimethylsilyl)oxy)methyl)-4-(4-
methoxyphenyl)oxazolidin-2-one (8):
To the solution of the compound 4 (0.2 g, 0.896 mmol) in dichloromethane (5 mL) was added
imidazole (0.121 g, 1.793 mmol), TBSCl (0.163 g, 1.075 mmol) and DMAP (cat) at 0 ºC and
stirred at room temperature for 1 h. The reaction mixture was then diluted with water and
extracted with dichloromethane. The organic layer was concentrated under reduced pressure
and purified through column chromatography (ethyl acetate/hexane, 1:1) to afford the
compound 8 (0.28 g, 94%) as colourless oil. [α] 30D = -44.4 (c=0.6, CHCl3); FT-IR (neat) vmax
1
cm-1 3274, 2930, 2856, 1747, 1611, 1513, 1464, 1248, 1177, 1076, 833, 775, 664. H NMR
(CDCl3, 500 MHz) : δ 7.18 (d, 2H, J = 8.6 Hz), 6.88 (d, 2H, J = 8.6 Hz), 5.43 (bs, 1H), 4.93
(d, 1H, J = 8.2 Hz), 4.82 (m, 1H), 3.81 (s, 3H), 3.48 (dd, 1H, J = 5.9 Hz, 10.9 Hz), 3.27 (dd,
1H, J = 6.1 Hz, 10.9 Hz), 0.80 (s, 9H), -0.08 (s, 3H), -0.13 (s, 3H). 13C NMR (CDCl3, 125
MHz): δ 159.8, 159.1, 128.3, 127.9, 114.0, 80.4, 61.4, 57.9, 55.3, 25.7, 18.1, -5.6, -5.7.
+
ESIMS m/z: [M+H] 338, HRMS (ESI) Calcd for C17
338.18.
H28
O
NSi [M+H]+ 338.18 found
4
4.4.
Synthesis
of.
(4R,5R)-3-(3-fluoro-4-morpholinophenyl)-5-(hydroxymethyl)-4-(4-
methoxyphenyl)oxazolidin-2-one (9):
To a solution of compound 8 (0.2 g, 0.593 mmol) in 1,4-dioxane (5 mL) was added
compound 6 (0.18 g, 0.593 mmol), CuI (0.005 g, 0.029 mmol, 5.0 mol %), and Cs2CO3
(0.385 g, 1.186 mmol) evacuated, and backfilled with argon then N,N’-
dimethylethylenediamine (cat) was added under argon. The reaction mixture was stirred at
110 °C for 3 h. The resulting bright yellow suspension was allowed to reach room
temperature and then filtered through a pad of celite eluting with ethyl acetate (2
X 10
mL).The filtrate was concentrated. The residue was dissolved in dry tetrahydrofuran (5 mL)
was added 1M. solution of TBAF (1.186 mL, 1.186 mmol) and stirred for 2 h at room
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temperature. The reaction mixture was then diluted with water and extracted with ethyl
acetate. The organic layer was concentrated under reduced pressure and purified through
column chromatography (ethyl acetate/hexane, 1:1) to afford the compound 9 (0.19 g, 80%
for two steps) as colourless oil. [α] 30D = -20.6 (c=0.26, CHCl3); FT-IR (neat) vmax cm-1 3395,
2927, 2855, 1737, 1611, 1513, 1447, 1378, 1249, 1176, 1047, 908, 847, 752, 664. 1H NMR
(CDCl3, 500 MHz) : δ 7.31 (dd, 1H, J = 14.3 Hz, 2.5 Hz,), 7.14 (d, 2H, J = 8.5 Hz), 7.00 (ddd,
1H, J = 8.8 Hz, 2.5 Hz, 1.0 Hz), 6.88 (d, 2H, J = 8.8 Hz), 6.78 (t, 1H, J = 9.1 Hz), 5.35 (d,
1H, J = 8.3 Hz), 4.92 (ddd, 1H, J = 12.3 Hz, 7.6 Hz, 4.5 Hz), 3.84-3.80 (m, 4H), 3.79 (s, 3H),
3.44 (m, 1H), 3.31 (m, 2H), 3.0-2.96 (m, 4H). 13C NMR (CDCl3, 125 MHz): δ 160.0, 155.2
(d, J = 247.0 Hz), 154.8, 136.5 (d, J = 8.1 Hz), 131.9 (d, J = 10.8), 128.1, 125.3, 118.5 (d, J =
3.6 Hz), 115.9 (d, J = 2.7 Hz), 114.6, 109.2 (d, J = 26.3 Hz), 77.5, 77.3, 66.9, 61.9, 55.2,
50.8. ESIMS m/z: [M+H]+ 403, HRMS (ESI) Calcd for C21H24O5N2F [M+H]+ 403.16561
found 403.16569.
4.5. Synthesis of. 3-(((4R,5R)-3-(3-fluoro-4-morpholinophenyl)-4-(4-methoxyphenyl)-2-
oxooxazolidin-5-yl)methoxy)-3-oxopropyl acrylate (10):
To a stirred solution of compound 9 (0.04 g, 0.099 mmol) in dry dichloromethane (5 mL)
were added acrylic acid (0.014 g, 0.199 mmol), DCC (0.044g, 0.199 mmol) and DMAP (cat)
at 0 ºC. The reaction mixture was allowed to room temperature and stirred for 1 h. The
reaction was quenched with saturated aq. NaHCO3 solution and the two resulting layers were
separated. The aqueous layer was extracted with dichloromethane (2 X 50 mL) and the
combined organic layers were washed with water and brine, dried over anhydrous Na2SO4.
The solvent was removed on a rotary evaporator and the residue was purified by column
chromatography (ethyl acetate/hexane, 3:7) to give the compound 10 (0.045 g, 87%) as
30
colourless oil. [α]
= -82.8 (c=0.27, CHCl3); FT-IR(neat) vmax cm-1 2924, 2854, 1741,
D
1612, 1513, 1449, 1404, 1334, 1251, 1173, 1030, 939, 810, 764. 1H NMR (CDCl3, 300 MHz)
: δ 7.31 (dd, 1H, J = 14.3Hz, 2.6 Hz), 7.14 (d, 2H, J = 8.6 Hz), 7.01 (dd, 1H, J = 8.8 Hz, 1.5
Hz), 6.88 (d, 2H, J = 8.4 Hz), 6.78 (t, 1H, J = 9.0 Hz), 6.41 (dd, 1H, J = 17.3 Hz, 1.3 Hz),
6.11 (dd, 1H, J = 17.3 Hz, 10.3 Hz), 5.85 (dd, 1H, J = 10.3 Hz, 1.1 Hz), 5.34 (d, 1H, J = 8.1
Hz), 5.01 (ddd, 1H, J = 12.8 Hz, 8.4 Hz, 3.9 Hz, ), 4.39 (t, 2H, J = 6.2 Hz), 3.93 (dd, 1H, J =
12.2 Hz, 3.7 Hz), 3.85-3.77 (m, 5H), 3.79 (s, 3H), 3.02-2.95 (m, 4H), 2.68 (t, 2H, J = 6.2 Hz).
13C NMR (CDCl3, 125 MHz): δ 169.9, 165.8, 160.2, 155.2 (d, J = 246.1 Hz), 154.5, 136.6 (d,
J = 8.1 Hz), 131.7 (d, J = 9.9 Hz), 131.3, 128.1, 127.9, 124.8, 118.5 (d, J = 4.5 Hz), 115.9 (d,
J = 2.7 Hz), 114.7, 109.1 (d, J = 25.4 Hz), 74.6, 66.8, 63.2, 61.9, 59.5, 55.2, 50.81, 50.80,
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33.5. ESIMS m/z: [M+H]+ 529, HRMS (ESI) Calcd for C27H30O8N2F [M+H]+ 529.19676
found 529.19725.
4.6. Synthesis of ((4R,5R)-3-(3-fluoro-4-morpholinophenyl)-4-(4-methoxyphenyl)-2-oxooxazolidin-5-
yl)methyl benzoate (11):
To the solution of the compound 9 (0.05 g, 0.098 mmol) in dichloromethane (10 mL) was
added triethylamine (0.04 mL, 0.296 mmol), benzoic anhydride (0.042 g, 0.186 mmol) and
DMAP (cat) at 0 ºC and then allowed to keep the reaction mixture at room temperature for 1
h. The reaction mixture was then diluted with water and extracted with dichloromethane. The
organic layer was concentrated under reduced pressure and purified through column
chromatography (ethyl acetate/hexane, 3:7) to afford the compound 11 (0.054 g, 90%) as
30
colourless oil. [α]
= -60.3 (c=0.19, CHCl3); FT-IR (neat) vmax cm-1 2922, 2852, 1753,
D
1
1724, 1514, 1449, 1395, 1269, 1251, 1176, 1116, 1028, 770, 713. H NMR (CDCl3, 500
MHz) : δ 7.99 (d, 2H, J = 7.3 Hz), 7.58 (t, 1H, J = 7.3 Hz), 7.44 (t, 2H, J = 7.6 Hz), 7.34 (dd,
1H, J = 14.3 Hz, 2.5 Hz), 7.18 (d, 2H, J = 8.5 Hz), 7.03 (dd, 1H, J = 8.6 Hz, 1.9 Hz), 6.87 (d,
2H, J = 8.5 Hz), 6.79 (t, 1H, J = 9.1 Hz), 5.41 (d, 1H, J = 8.2 Hz), 5.16 (td, 1H, J = 3.8 Hz,
3.9 Hz, 4.1 Hz), 4.17 (dd, 1H, J = 12.3 Hz, 3.9 Hz), 3.98 (dd, 1H, J = 12.3 Hz, 7.9 Hz), 3.85-
13
3.80 (m, 4H), 3.77 (s, 3H), 3.01-2.97 (m, 4H). C NMR (CDCl3, 125 MHz): δ 166.6, 160.6,
155.2 (d, J = 247.9 Hz), 154.2, 134.5 (d, J = 9.9 Hz), 133.4, 131.5 (d, J = 15.4 Hz), 129.7,
129.1, 128.4, 128.1, 127.6, 116.3 (d, J = 3.6 Hz), 116.0 (d, J = 3.6 Hz), 114.7, 109.8 (d, J =
24.5 Hz), 74.9, 66.9, 63.2, 62.1, 55.2, 50.8. ESIMS m/z: [M+H]+ 507, HRMS (ESI) Calcd for
C28H28O6N2F [M+H]+ 507.19 found 507.19.
4.7.
Synthesis
of
(4R,5R)-3-(3-fluoro-4-morpholinophenyl)-4-(4-methoxyphenyl)-5-
vinyloxazolidin-2-one (13):
To a solution of compound 6 (0.8 g, 3.65 mmol) in 1,4-dioxane (10 mL) was added
compound 12 (1.12 g, 3.65 mmol), CuI (0.034 g, 0.182 mmol, 5.0 mol %), and Cs2CO3 (2.37
g, 7.30 mmol) evacuated for a short while and backfilled with argon then N,N’-
dimethylethylenediamine (10 mol%) was added under argon. The reaction mixture was
stirred at 110 °C for 3 h. The resulting bright yellow suspension was allowed to reach room
temperature and then filtered through a pad of celite eluting with ethyl acetate (2 X 20 mL).
The filtrate was concentrated and the residue was purified by silica gel column
chromatography (ethyl acetate/hexane, 7:3) to afford the pure compound 13 (1.308 g, 90%)
30
as a light brown syrup. [α]
= -8.18 (c=2.4, CHCl3); FT-IR(neat) vmax cm-1 2928, 2854,
D
9

ACCEPTED MANUSCRIPT
1753, 1612, 1513, 1375, 1249, 1227, 1175, 1116, 1030, 938, 833, 772. 1H NMR (CDCl3, 500
MHz) : δ 7.21-7.14 (m, 3H), 6.95 (dd, 1H, J = 8.8 Hz, 2.2Hz), 6.87 (d, 1H, J = 8.6 Hz), 6.77
(t, 1H, J = 9.0 Hz), 6.01 (m, 1H), 5.43-5.36 (m, 2H), 4.88 (d, 1H, J = 6.8 Hz), 4.70 (m, 1H),
3.83-3.79 (m, 4H), 3.78 (s, 3H), 3.00-2.95 (m, 4H). 13C NMR (CDCl3, 125 MHz): δ 159.9,
155.2, 155.1 (d, J = 246.1 Hz), 136.8 (d, J = 8.1 Hz), 132.9, 131.7 (d, J = 9.9 Hz), 128.4,
127.7, 119.9, 118.4 (d, J = 3.6 Hz), 117.1 (d, J = 2.7 Hz), 114.6, 110.1 (d, J = 25.4 Hz), 82.4,
66.8, 66.1, 55.2, 50.77, 50.74. ESIMS m/z: [M+H]+ 399, HRMS (ESI) Calcd for
C22H24O4N2F [M+H]+ 399.1714 found 399.1713.
4.8.
Synthesis
of
(4R,5S)-3-(3-fluoro-4-morpholinophenyl)-5-(hydroxymethyl)-4-(4-
methoxyphenyl)oxazolidin-2-one (14):
To a solution of compound 13 (0.5 g, 1.256 mmol) in dry dichloromethane (10 mL) at –78 °C
ozone gas was passed for 30 minutes (until solution turns to light blue color). The reaction
mixture was treated with dimethyl sulphide then concentrated under reduced pressure. The
crude reaction mixture dissolved in methanol (5 mL), to it was added NaBH4 (0.19 g, 5.025
mmol) at 0 ºC and stirred the reaction mixture for 2 h at 0 °C. Then the reaction mixture was
quenched with saturated NH4Cl solution and solvents were removed under reduced pressure.
Crude compound was partitioned between dichloromethane and water. Aqueous layer was
again extracted with dichloromethane. Combined organic layers were dried over Na2SO4 and
the solvent was removed under reduced pressure followed by silica gel column
chromatography (ethyl acetate/hexane, 7:3) to afford the pure compound 14 (0.439 g, 87%) as
colourless oil. [α] 30D = -15.7 (c=2.1, CHCl3); FT-IR (neat) vmax cm-1 3395, 3013, 2960, 2836,
1
1741, 1612, 1512, 1377, 1227, 1175, 1032, 904, 745, 664. H NMR (CDCl3, 300 MHz) : δ
7.25-7.15 (m, 3H), 6.98 (d, 1H, J = 8.8 Hz), 6.88 (d, 2H, J = 8.4 Hz), 6.78 (t, 1H, J = 9.0 Hz),
5.21 (d, 1H, J = 6.6 Hz), 4.38 (m, 1H), 4.01 (m, 1H), 3.85-3.72 (m, 5H), 3.78 (s, 3H), 3.02-
2.95 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ 159.8, 155.6, 155.1 (d, J = 245.8 Hz), 136.9 (d, J
= 8.7 Hz), 131.5 (d, J = 9.8 Hz), 129.3, 127.8, 118.4 (d, J = 3.8 Hz), 117.4 (d, J = 2.7 Hz),
114.7, 110.4 (d, J = 25.2 Hz), 82.0, 66.8, 61.3, 61.2, 55.2, 50.77, 50.74. ESIMS m/z: [M+H]+
403, HRMS (ESI) Calcd for C21H24O5N2F [M+H]+ 403.16638 found 403.16689.
4.9.
Synthesis
of
((4R,5S)-3-(3-fluoro-4-morpholinophenyl)-4-(4-methoxyphenyl)-2-
oxooxazolidin-5-yl)methyl benzoate (15):
To the solution of the compound 14 (0.05 g, 0.098 mmol) in dichloromethane (10 mL) was
added triethylamine (0.04 mL, 0.296 mmol), benzoic anhydride (0.042 g, 0.186 mmol) and
10

ACCEPTED MANUSCRIPT
DMAP (cat) at 0 ºC and then allowed to keep the reaction mixture at room temperature for 1
h. The reaction mixture was then diluted with water and extracted with dichloromethane. The
organic layer was concentrated under reduced pressure and purified through column
chromatography (ethyl acetate/hexane, 3:7) to give the compound 15 (0.047 g, 77%) as
30
colourless oil. [α]
= +35.02 (c=2.2, CHCl3); FT-IR (neat) vmax cm-1 2959, 2836, 1754,
D
1
1723, 1611, 1513, 1449, 1377, 1268, 1249, 1116, 1028, 772, 712. H NMR (CDCl3, 500
MHz) : δ 7.99 (d, 2H, J = 8.0 Hz), 7.58 (t, 1H, J = 7.47 Hz), 7.42 (t, 2H, J = 7.7 Hz), 7.25-
7.19 (m, 3H), 6.97 (dd, 1H, J = 8.6 Hz, 2.5 Hz), 6.89 (d, 2H, J = 8.6 Hz), 6.77 (t, 1H, J = 9.0
Hz), 5.08 (d, 1H, J = 5.0 Hz), 4.68-4.60 (m, 3H), 3.83-3.80 (m, 4H), 3.79 (s, 3H), 3.00-2.97
13
(m, 4H). C NMR (CDCl3, 125 MHz): δ 166.0, 160.1, 155.1 (d, J = 247.0 Hz), 154.8, 136.9
(d, J = 9.0 Hz), 133.5, 131.5 (d, J = 9.9 Hz), 129.7, 129.1, 128.9, 128.5, 127.5, 118.5 (d, J =
3.6 Hz), 116.9 (d, J = 3.6 Hz), 114.8, 110.0 (d, J = 25.4 Hz), 79.1, 66.8, 63.7, 62.6, 55.2,
+
50.77, 50.74. ESIMS m/z: [M+H] 507, HRMS (ESI) Calcd for C28H28O6N2F [M+H]+
507.19259 found 507.1926.
4.10. Synthesis of 3-(((4R,5S)-3-(3-fluoro-4-morpholinophenyl)-4-(4-methoxyphenyl)-2-
oxooxazolidin-5-yl)methoxy)-3-oxopropyl acrylate (16):
To a stirred solution of compound 14 (0.04 g, 0.099 mmol) in dry dichloromethane (5 mL)
were added acrylic acid (0.014 g, 0.199 mmol), DCC (0.044 g, 0.199 mmol) and DMAP (cat)
at 0 ºC. The reaction mixture was allowed to room temperature and stirred for 1 h. The
reaction was quenched with saturated aq. NaHCO3 solution and the two resulting layers were
separated. The aqueous layer was extracted with dichloromethane (2 X 50 mL) and the
combined organic layers were washed with water and brine, dried over anhydrous Na2SO4.
The solvent was removed on a rotary evaporator and the residue was purified by column
chromatography (ethyl acetate/hexane, 3:7) to afford the compound 16 (0.044 g, 85%) as
colourless oil. [α] 30D = -0.4 (c=1.8, CHCl3); FT-IR (neat) vmax cm-1 2928, 2855, 1743, 1613,
1
1513, 1448, 1378, 1248, 1169, 1029, 938, 809, 747, 664. H NMR (CDCl3, 500 MHz) : δ
7.23-7.17 (m, 3H), 6.97 (ddd, 1H, J = 8.8 Hz, 2.5 Hz, 1.0 Hz), 6.89 (d, 2H, J = 8.6 Hz), 6.79
(t, 1H, J = 9.0 Hz), 6.40 (dd, 1H, J = 17.3 Hz, 1.3 Hz), 6.09 (dd, 1H, J = 17.3 Hz, 10.3 Hz),
5.84 (dd, 1H, J = 10.3 Hz, 1.3 Hz), 5.02 (d, 1H, J = 6.1 Hz), 4.53 (m, 1H), 4.48-4.38 (m, 4H),
3.83-3.80 (m, 4H), 3.79 (s, 3H), 3.01-2.97 (m, 4H), 2.75 (t, 2H, J = 6.1 Hz). 13C NMR
(CDCl3, 75 MHz): δ 170.2, 165.7, 160.1, 155.1 (d, J = 246.9 Hz), 154.7, 137.0 (d, J = 8.7
Hz), 131.4, 131.3(d, J = 9.9 Hz), 128.8, 127.8, 127.6, 118.5 (d, J = 3.8 Hz), 117.0 (d, J = 2.7
Hz), 114.8, 110.1 (d, J = 25.2 Hz), 78.8, 66.8, 63.2, 62.3, 59.5, 55.2, 50.77, 50.74, 33.7.
11

ACCEPTED MANUSCRIPT
ESIMS m/z: [M+H] + 529, HRMS (ESI) Calcd for C27H30O8N2F [M+H]+ 529.19807 found
529.19852.
4.11. Synthesis of (4R,5R)-5-ethyl-3-(3-fluoro-4-morpholinophenyl)-4-(4-methoxyphenyl)
oxazolidin-2-one (17):
To a solution of compound 13 (0.06 g, 0.15 mmol) in methanol (2 mL) was added 10 % Pd/C
(cat) and stirred the reaction mixture under H2 atmosphere for 1 h. The reaction mixture was
filtered through a pad of celite and concentrated under reduced pressure. The residue was
purified by column chromatography (ethyl acetate/hexane, 1:4) to afford the pure compound
30
17 (0.051 g, 85%) as a colourless syrup. [α]
= - 37.41(c=2.05, CHCl3); FT-IR(neat) vmax
D
cm-1 2964, 2934, 2854, 1746, 1612, 1512, 1446, 1376, 1337, 1248, 1175, 1116, 1031, 938,
1
775, 730, 665. H NMR (CDCl3, 500 MHz) : δ 7.22-7.16 (m, 3H), 6.97 (dd, 1H, J = 8.8 Hz,
1.6 Hz), 6.87 (d, 2H, J = 8.6 Hz), 6.77 (t, 1H, J = 9.0 Hz), 4.80 (d, 1H, J = 6.2 Hz), 4.25 (m,
1H), 3.83-3.79 (m, 4H), 3.78 (s, 3H), 3.00-2.96 (m, 4H), 1.90-1.83 (m, 2H), 1.07 (t, 3H, J =
7.3 Hz). 13C NMR (CDCl3, 75 MHz): δ 159.8, 155.4, 155.1 (d, J = 246.4 Hz), 136.6 (d, J =
8.8 Hz), 132.0 (d, J = 9.9 Hz), 129.6, 127.7, 118.4 (d, J = 3.8 Hz), 117.0 (d, J = 2.7 Hz),
114.7, 110.0 (d, J = 25.3 Hz), 83.3, 66.8, 65.7, 55.2, 50.85, 50.80, 27.1, 9.0. ESIMS m/z:
[M+H]+ 401, HRMS (ESI) Calcd for C22H26O4N2F [M+H]+ 401.1871 found 401.1876.
4.12. Synthesis of (4R,5R)-3-(3-fluoro-4-morpholinophenyl)-5-(2-hydroxyethyl)-4-(4-
methoxyphenyl)oxazolidin-2-one (18):
To a solution of compound 13 (0.06 g, 0.15 mmol) in dry tetrahydrofuran (10 mL) was added
°
BH3.S(CH3)2 complex (0.024 mL, 0.301 mmol) at 0 C under nitrogen. The mixture was
allowed to warm to room temperature over 3 h and then evaporated under reduced pressure.
sodium hydroxide solution (3M, 0.75 mmol) and 30% aqueous H2O2 (0.75 mmol) were added
°
successively to a solution of the residue in tetrahydrofuran (10 mL) at 0 C. The turbid
solution was warmed to room temperature over 2 h and then quenched with water (20 mL)
and extracted with ethyl acetate (3
x
10 mL). The ethyl acetate extracts were dried (Na2SO4),
and then concentrated in vacuo. The residue was purified by column chromatography (ethyl
30
acetate/hexane, 1:1) to give compound 18 (0.047 g, 75%) as colourless oil. [α]
= -18.79
D
(c= 0.36, CHCl3); FT-IR(neat) vmax cm-1 3432, 2925, 2855, 1741, 1612, 1513,1446, 1378,
1248, 1229, 1175, 1113, 1030, 835, 750, 664. 1H NMR (CDCl3, 500 MHz): δ 7.21 (d, 2H, J =
8.6 Hz), 7.17 (dd, 1H, J = 14.1 Hz, 2.5 Hz), 6.96 (ddd, 1H, J = 8.6 Hz, 2.5 Hz, 1.0 Hz), 6.87
12

ACCEPTED MANUSCRIPT
(d, 2H, J = 8.6 Hz), 6.77 (t, 1H, J = 9.0 Hz), 4.95 (d, 1H, J = 6.4 Hz), 4.51 (m, 1H), 3.87 (m,
2H), 3.83-3.77 (m, 5H), 3.78 (s, 3H), 3.0-2.96 (m, 4H), 2.08 (m, 2H). 13C NMR (CDCl3, 75
MHz): δ 159.9, 155.3, 155.1 (d, J = 246.1 Hz), 136.8 (d, J = 9.0 Hz), 131.8 (d, J = 9.9 Hz),
129.1, 127.8, 118.4 (d, J = 4.5 Hz), 117.2 (d, J = 3.6 Hz), 114.7, 110.2 (d, J = 25.4 Hz), 79.8,
66.8, 66.1, 58.5, 55.2, 50.8, 50.7, 36.2. ESIMS m/z: [M+H]+ 417, HRMS (ESI) Calcd for
C22H26O5N2F [M+H] + 417.1801 found 417.1803.
4.13. Synthesis of (4R, 5R)-5-(azidomethyl)-3-(3-fluoro-4-morpholinophenyl)-4-(4-
methoxyphenyl)oxazolidin-2-one (19):
To the solution of the compound 14 (0.19 g, 0.472 mmol) in dichloromethane (10 mL) was
added triethylamine (0.196 mL, 1.417 mmol), methanesulfonyl chloride (0.054 ml, 0.708
mmol) and DMAP (cat) at 0 °C and then allowed to keep the reaction mixture at room
temperature for 1h. The reaction mixture was then diluted with water and extracted with
dichloromethane. The residue was dissolved in dry N,N-dimethylformamide (4 mL) and
°
sodium azide (0.403 g, 0.945 mmol) was added. The reaction mixture was heated at 100 C
for 2 h. The reaction mixture was then allowed to room temperature, diluted with water and
extracted with dichloromethane (2 X 10 mL) and the combined organic layers were washed
with water and brine, dried over anhydrous Na2SO4. The solvent was removed on a rotary
evaporator and the residue was purified by column chromatography (ethyl acetate/hexane,
30
3:7) to give compound 19 (0.173 g, 86% for two steps) as yellow oil. [α]
= -32 (c= 0.9,
D
CHCl3); FT-IR(neat) vmax cm-1 2921, 2853, 2106, 1754, 1612, 1513, 1444, 1378, 1249, 1116,
1030, 938, 752, 665. 1H NMR (CDCl3, 500 MHz) : δ 7.23-7.18 (m, 3H), 6.98 (dd, 1H, J = 8.6
Hz, 1.6 Hz), 6.88 (d, 2H, J = 8.6 Hz), 6.79 (t, 1H, J = 9.1 Hz), 5.04 (d, 1H, J=5.9 Hz), 4.44
(m, 1H), 3.83-3.80 (m, 4H), 3.78 (s, 3H), 3.74 (dd, 1H, J = 13.4 Hz, 4.1 Hz), 3.60 (dd, 1H, J
= 13.4 Hz, 4.1 Hz), 3.01-2.97 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ 160.0
, 155.1 (d, J =
246.9 Hz), 154.5, 137.0 (d, J = 8.7 Hz), 131.3 (d, J = 9.8 Hz), 128.8, 127.6, 118.5 (d, J = 3.8
Hz), 117.2 (d, J = 2.7 Hz), 114.8, 110.2 (d, J = 25.2 Hz), 79.6, 66.8, 62.6, 55.2, 51.8, 50.76,
50.73, 29.6. ESIMS m/z: 428[M+H]+, HRMS (ESI) Calcd for C21H23O4NF [M+H]+
428.17286 found 428.17229.
4.14.
Synthesis
of
(4R,5R)-5-(aminomethyl)-3-(3-fluoro-4-morpholinophenyl)-4-(4-
methoxyphenyl)oxazolidin-2-one (20):
To a solution of compound 19 (0.15 g, 0.351 mmol) in methanol (5 mL) was added 10 %
Pd/C (cat) and stirred the reaction mixture under H2 atmosphere for 3 h. The reaction mixture
13

ACCEPTED MANUSCRIPT
was filtered through celite and concentrated under reduced pressure. The crude was purified
by column chromatography (chloroform/methanol, 9:1) to afford the compound 20 (0.102 g,
30
73%) as colourless oil. [α]
= +3.0 (c=2.5, CHCl3); FT-IR(neat) vmax cm-1 2959, 2835,
D
1
1749, 1612, 1513, 1445, 1378, 1334, 1229, 1115, 1030, 905, 833, 772, 729, 665. H NMR
(CDCl3, 500 MHz) : δ 7.27-7.17 (m, 3H), 6.97 (dd, 1H, J = 8.6 Hz, 2.5 Hz), 6.86 (d, 2H, J =
8.6 Hz), 6.77 (t, 1H, J = 9.1 Hz), 5.06 (d, 1H, J = 6.4 Hz), 4.34 (m, 1H), 4.21 (m, 1H), 3.83-
3.78 (m, 4H), 3.77 (s, 3H), 3.01-2.95 (m, 4H). 13C NMR (CDCl3, 75 MHz): δ 159.8, 155.2,
155.1 (d, J = 246.4 Hz), 136.8, 131.6 (d, J = 9.8 Hz), 129.3, 127.8, 118.4 (d, J = 3.8 Hz),
117.2 (d, J = 2.7 Hz), 114.7, 110.2 (d, J = 25.2 Hz), 82.5, 66.8, 62.7, 55.2, 50.7, 43.3. ESIMS
m/z: [M+H]+ 402, HRMS (ESI) Calcd for C21H25O4N3F [M+H] + 402.18236 found 402.1817.
4.15. Synthesis of N-(((4R,5R)-3-(3-fluoro-4-morpholinophenyl)-4-(4-methoxyphenyl)-2-
oxooxazolidin-5-yl)methyl)acetamide (21):
To the solution of the compound 20 (0.06 g, 0.149 mmol) in dichloromethane (5 mL) was
added triethylamine (0.062 mL, 0.448 mmol), acetic anhydride (0.022 g, 0.224 mmol) and
DMAP (cat) at 0 ºC and then allowed to keep the reaction mixture at room temperature for 1
h. The reaction mixture was then diluted with water and extracted with dichloromethane. The
organic layer was concentrated under reduced pressure and purified through column
chromatography (ethyl acetate/hexane, 3:7) to afford the compound 21 (0.056 g, 85%) as
colourless syrup. [α] 30D = -26.7 (c=1, CHCl3); FT-IR (neat) vmax cm-1 3323, 2961, 2835,
1750, 1659, 1612, 1513, 1376, 1248, 1176, 1114, 1031, 914, 772, 729, 665. 1H NMR (CDCl3,
500 MHz) : δ 7.24 (d, 2H, J = 8.6 Hz), 7.18 (dd, 1H, J = 14.1 Hz, 2.5 Hz), 6.92 (dd, 1H, J =
8.8 Hz, 1.8 Hz), 6.86 (d, 2H, J = 8.6 Hz), 6.77 (t, 1H, J = 9.0 Hz), 6.08 (t, 1H, J = 6.1 Hz),
5.01 (d, 1H, J = 6.5 Hz), 3.85 (m, 1H), 3.83-3.80 (m, 4H), 3.78 (s, 3H), 3.61-3.55 (m, 1H),
3.00-2.96 (m, 4H), 2.04 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 171.0, 159.9, 155.9 (d, J =
246.4 Hz), 155.1, 136.9 (d, J = 8.8 Hz), 132.6 (d, J = 8.2 Hz), 128.6, 127.8, 118.4 (d, J = 3.2
Hz), 117.1 (d, J = 2.7 Hz), 114.7, 110.2 (d, J = 25.2 Hz), 80.7, 66.8, 62.5, 55.2, 50.78, 50.74,
40.1, 23.2. ESIMS m/z: 444 [M+H]+, HRMS (ESI) Calcd for C23H27O5N3F [M+H]+
444.19293 found 444.19259.
4.16. Cell Based Assays
4.16.1. Cytotoxicity evaluation against different cancer cell lines
14

ACCEPTED MANUSCRIPT
Cellular viability in presence of the compound was tested by performing a Sulforhodamine B
Assay (SRB) [18]. Briefly, the cell line of interest was seeded in flat bottom 96- well plate
(5000 cells/100 µL) in a medium containing 10% serum followed by incubating the plate for
18-20 h in an incubator continuously supplied with 5 % CO2, allowing cells to adhere to the
surface of the wells. After 18 h the cells were treated with the compound at desired
concentration. Working dilutions of 500 µM concentration of the compounds were prepared,
of which 2 µl aliquot was added to the each well, thereby making the final concentration of
compound 0 to 100 µM. DMSO and doxorubicin (as standard control anticancer drug) were
taken as vehicle and positive controls respectively. Further, the plates were incubated for
another 48 h in an incubator maintained at 37 ºC with a constant supply of 5% CO2. After 48
h of treatment cells were fixed using 10% TCA for 1 h at 4 ºC. The plate was rinsed carefully
with MQ water removing TCA and air dried. Then 0.057% SRB solution was added to plates
and incubated for 30 minutes before washing to remove unbound SRB using 1% acetic acid.
The plates were then air dried and the absorbance was measured using Perkin –Elmer
Multimode Reader at 510 nm. To measure the absorbance, 100 µL of 10 mM Tris Base was
added to each well to solubilise the SRB. The measure of absorbance is directly proportional
to cell growth and is thus used to calculate the IC50 values. In the present study, four types of
cancer cell lines i.e. lung cancer (A549); breast cancer (MCF-7), prostate cancer (DU145)
and cervical cancer (HeLa) cell lines were tested for the cytotoxic effect of the series of
compounds. Based on the IC50 values obtained, the compound 17 was selected for further
assays to ascertain its effect on prostate cancer cell line (DU145).
4.16.2. Change in Morphology
As the compound 17 was found to be cytotoxic to DU145 cells, its effect on the morphology
of cells was further ascertained. A 24 well plate was seeded with cells in a manner previously
described and incubated for 18-20 h. Then the cells were treated with increasing
concentrations of 17. After another 48 h of incubation, the experiment was terminated and the
cells were observed under the microscope and images were captured using Olympus Xi71
microscope.
4.16.3. Colony formation assay
The long term effect of 17 on the anchorage-independent growth of DU145 cells was
analyzed by soft agar assays as reported previously with minor modifications [17]. In the soft
agar assay, base agar was prepared by mixing 1% of agarose (Bacto Agar: Becton,
Dickinson, Sparks, MD) with 2 × DMEM with 20% FBS and 2X antibiotics in 6-well plates
15

ACCEPTED MANUSCRIPT
in order to achieve final concentration of 0.5% of agar in 1X growth medium with 10%
serum concentration. After the solidification of the base agar, 2.5 × 104 cells were mixed with
cultivation medium containing compound at varying concentrations and agar solution to
obtain a final concentration of 0.35% agar. This was spread on top of the base agar previously
solidified. The plate was incubated for 9 days with periodic replenishment every 3 days with
medium and compound. Over period of time plates were monitored regularly for appearance
of colonies. After 9 days of incubation the plates were stained with 0.005% crystal violet
solution until colonies turned purple in colour. The excessive stain was washed off using MQ
water and the colonies were photographed and counted using a microscope.
4.16.4. Determination of caspase-3 and -9 activities
Caspase-3 and -9 activities were measured by treating the DU145 cells with 17 using specific
fluorogenic substrates Ac-DEVD-AMC and Ac-LEHD-AFC. DMSO and doxorubicin were
taken as negative and positive controls respectively. After 48 h treatment of cells with 17,
harvested cells were lysed directly in caspase lysis buffer (50 mM HEPES, 5mM CHAPS, 5
mM DTT, pH 7.5). The lysates were incubated with the respective substrate (Ac-DEVD-
AFC/Ac-LEHD-AMC) in 20mM HEPES (pH 7.5), 0.1% CHAPS, 2mM EDTA and 5 mM
DTT at 37 °C for 2 h. The release of AFC and AMC was analyzed by a fluorometer using an
excitation/emission wavelength of 400/505 nm (for AFC) and 380/460 nm (for AMC) which
is directly proportional to caspase-9 and-3 activity respectively. The observed fluorescence
values were normalized with total protein concentration estimated by Bradford method and
the relative caspase activities were calculated as the ratio of values between mock treated
(DMSO) and treated cells.
4.16.5. Senescence Assay
Small molecules displaying anticancer potential may induce senescence in cancer cells
preventing their proliferation [17]. Thus, we have studied the ability of 17 to induce
senescence in healthy DU145 cells. Induction of cellular senescence was monitored by
measuring senescence-associated β-galactosidase (SA-βgal) activity (pH 6.0) assay in DU145
cells exposed to 17. A 24 well plate was seeded with cells as previously described and treated
with the compound and subsequently incubated for 48 h. After 48 h, the media was aspirated
and the cells were washed with PBS (2 × 1 min). Cells were fixed by adding enough volume
of fixation solution (2% formaldehyde and 0.2% glutaraldehyde in PBS solution) to
submerge the cells in solution. After incubation for 5 minutes at room temperature the
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fixation solution was removed and the cells were washed twice with PBS (2 × 1 min). Then
the fixed cells were stained with freshly prepared staining solution (40 mM citric acid/Na
phosphate buffer, 5 mM K4[Fe (CN) 6]3H2O, 5 mM K3[Fe (CN) 6], 150 mM sodium chloride,
2 mM magnesium chloride and 1 mg ml − 1 X-gal in distilled water) for overnight at 37 °C.
The excess stain was removed by repeated washings with PBS and plate was allowed for
drying at room temperature. The positively stained cells for SA-βgal levels were observed
and photographed under a Olympus Xi71 microscope.
4.16.6. PI uptake for analysis of cell death
Cell death induced by 17 was determined as a measure of PI uptake. Cells were harvested
after treatment with compound at desired concentration and fixed in 70% ethanol at -20 °C
for overnight. All cells in the form of a pellet re-suspended in PI solution (RNase -0.1
mg/mL, Triton X-100-0.05%, PI-50 ꢀg/mL) and incubated for 1 h in dark at room
temperature. The excess PI solution was washed away by repeated washings with PBS buffer.
The resultant PI uptake was analyzed by fluorescence activated cell sorting (FACS Calibur
System; BD Bio- science, Erembodegem, Belgium) in a FL-2 fluorescence detector (10000
events were recorded for each condition). Flow cytometry data was analyzed using FCS
express 4 software (De Novo Software, Los Angeles, CA).
4.16.7. Mitochondrial Membrane Potential assay:
The ability of 17 to induce apoptosis via caspase activation was analysed by determining the
caspase activities in the treated cells. Mitochondrion plays an important role in activating
these caspases. The detection of changes in mitochondrial inner membrane electrochemical
potential in living cells was done using the cationic, lipophilic dye, JC-1 (5, 5’, 6, 6’-
tetracholoro-1, 1’, 3, 3’-tetraethylbenzimidazolocarbocyanine iodide). In healthy cells the JC-
1 dye is concentrated in the mitochondrial matrix and form of red fluorescent aggregates (J-
aggregates). When the cells become apoptotic, the mitochondrial membrane potential gets
affected and the JC-1 dye is released into the cytosol in the form of monomers, leading to a
shift from red fluorescence (Rhodamine) to green fluorescence (FITC). In the assay
conducted, we observed whether compound 17 shows any effect on the mitochondrial
membrane. The DU145 cells were seeded in 24 well plate and incubated for 18 h at 37 °C
with constant supply of 5% CO2. When the cells had adhered to the surface of the well, they
were treated with either 17 or DMSO as control. After 24 h of post treatment with 17, the
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cells were stained with JC-1 dye (0.625% MilliQ water). The working dye solution was
prepared by mixing equal volumes of the stock dye complete growth medium. The media in
the wells was replaced with dye mix and incubated for 20 minutes at 37 °C in a humidified
incubator. After aspiration of staining solution cells were washed with growth medium for
two times and covered with growth medium. The resultant fluorescence was observed under
the microscope (Olympus Xi71, Japan).
Acknowledgments
A.N thanks UGC, and M.V.R thanks CSIR, New Delhi for research fellowship. We also
thank CSIR for financial support in the form of XII FYP Programme DENOVA (CSC0205)
and SMILE (CSC0111). The authors also thank Director, CSIR-IICT for the constant support
and encouragement.
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Figure Captions:
Table 1: Four representative cell lines were tested with the series of compounds to determine
their cytotoxicity. Table shows the IC50 values of the compounds against the cell lines.
Table 2: Compound 17 induced G0/G1 phase cell cycle arrest in DU145 cells. Cells were
treated with varying concentrations of 17 (5, 10, 15, 20 and 25 µM) for 48 h and cell cycle
progression was examined by flow cytometry. Table shows the percentage cell fractions in
G0/G1, S and G2/M phases of 17 treated DU145 cells.
Figure 1: Some of the active oxazolidinone derivatives.
Figure 2: Structure of linezolid 3 and new proposed structures (I, II)
Figure 3: DU145 cell were treated with compound 17 at indicated concentration or DMSO.
Upon exposure of DU145 cells to 17 the extent of change in cell morphology of cells is
observed with increasing concentration.
Figure 4: Long term effect of compound 17 on the number of colony-forming DU145 cells.
DU145 cells were treated with desired concentration of 17 (0−25 ꢀM) and allowed to grow
for 9 days to form colonies. Representative images of the colony-forming assay are shown
here. Number of colonies and their size formed by DU145 in soft agar is decreased on
exposure to 17
Figure 5: Cell cycle analysis of DU145 cells treated with 17. Cells were treated with either
DMSO or 17 and the DNA content was measured by propidium iodide staining. To determine
the distribution of cells in various phases of cell cycle was taken as control. The percentage
of cells increased in G0/G1 phase up on exposure to 17 suggesting its role in G0/G1 arrest.
Figure 6: Effect of 17 on activation of caspases inducing apoptosis in DU145 cells.
Treatment of DU145 cells with different concentration of 17 for 48 h induced activation of
caspases-3 (A) and caspase-9 (B) significantly in concentration dependent manner.
Doxorubicin was used as control for activation of both caspases. All experiments were
carried out in triplicates and mean values are presented here.
Figure 7: Effect of 17 on mitochondrial membrane potential inducing activation of caspase 9
was determined by staining the cells exposed to 17 with JC-1 dye. Cell treated with DMSO
show red fluorescence as the JC-1 dye is localized and aggregated in the mitochondrial
membrane. In cells exposed to 17, green fluorescence is increased where as red aggregated
21

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fluorescence is decreased indicating depolarization of mitochondrial membrane.
Figure 8: Senescence induced by compound 17 was quantified using SA-ß-gal-staining. SA-
ß-gal positive cells were indicated with arrow mark. The number of SA-ß-gal positive cells is
increased with increasing the concentration of compound indicates that compound 17
inducing senescence of DU145 cells.
Scheme 1: Retrosynthetic analysis of new proposed structures (I & II)
Scheme 2: Synthesis of key intermediate 6
Scheme 3: Synthesis of analogues 9, 10, 11
Scheme 4: Synthesis of analogues 5, 13, 14
Scheme 5: Synthesis of analogues 15, 16
Scheme 6: Synthesis of analogues 17, 18
Scheme 7: Synthesis of analogues 20, 21
22

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Table 1: Four representative cell lines were tested with the series of compounds to determine
their cytotoxicity. Table shows the IC50 values of the compounds against the cell lines.
DU145
IC50 Std. Dev IC50 Std. Dev IC50 Std. Dev IC50 Std. Dev
A549
HELA
MCF 7
Compound
No.
25.53
43.17
42.4
2.74
7.87
11.59
4.2
46.45
38.51
31.76
30.78
40.62
32.8
48.16
24.85
21.13
9.47
1.38
4.63
2.55
5.76
24.24
3.53
20.14
12.93
15.58
0.18
0.38
30.51
7.28
8.72
0.1
26.02
35.89
28.61
19.19
56.69
39.77
48.58
86.56
57.65
42.66
23.31
43.17
>100
51.27
10.89
3.3
12.04
7.58
0.7
25.69
2.15
17.55
25.33
2.69
5.84
2.04
8.86
0
82.42
36.11
61.54
69.42
>100
>100
69.96
>100
74.03
38.22
>100
>100
>100
>100
8.62
10.35
7.45
6.93
33.88
0
0
8.73
0
14.5
10.13
0
0
0
4
9
10
11
5
12
13
14
15
16
17
18
20
21
24.81
31.15
21.31
24.45
24.64
23.87
14.15
13.4
12.24
7.57
11.49
8.74
7.72
1.46
1.22
2.78
5.99
20.64
0.12
8.69
30.31
21.16
54.4
69.52
33.03
26.84
6.7
6.33
0.09
0
0.19
Doxorubicin
8.4
Table 2: Compound 17 induced G0/G1 phase cell cycle arrest in DU145 cells. Cells were
treated with varying concentrations of 17 (5, 10, 15, 20 and 25 µM) for 48 h and cell cycle
progression was examined by flow cytometry. Table shows the percentage cell fractions in
G0/G1, S and G2/M phases of 17 treated DU145 cells.
G0/G1
70.54
75.65
75.29
79.14
80.5
S
G2/M
23.09
19.45
20.49
16.7
5.77
4.61
3.85
3.56
1.93
0.79
DMSO
5 µM
10 µM
15 µM
20 µM
25 µM
16.19
6.54
91.11
23

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Figure 1: Some of the active oxazolidinone derivatives.
Figure 2: Structure of linezolid 3 and new proposed structures (I, II)
24

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DMSO
5µM
10µM
25µM
15µM
20µM
Figure 3: DU145 cell were treated with compound 17 at indicated concentration or DMSO.
Upon exposure of DU145 cells to 17 the extent of change in cell morphology of cells is
observed with increasing concentration.
DMSO
10µM
DMSO
5µM
Doxorubicin
15µM
25µM
Figure 4: Long term effect of compound 17 on the number of colony-forming DU145 cells.
DU145 cells were treated with desired concentration of 17 (0−25 ꢀM) and allowed to grow
for 9 days to form colonies. Representative images of the colony-forming assay are shown
here. Number of colonies and their size formed by DU145 in soft agar is decreased on
exposure to 17
25

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600
450
300
150
600
450
300
150
600
450
300
150
10µM
DMSO
5µM
0
0
0
0
64
128
FL 2 Area
192
256
0
64
128
FL 2 Area
192
256
0
64
128
FL 2 Area
192
256
600
600
600
20µM
15µM
25µM
450
300
150
0
450
300
150
0
450
300
150
0
0
64
128
FL 2 Area
192
256
0
64
128
FL 2 Area
192
256
0
64
128
FL 2 Area
192
256
Figure 5: Cell cycle analysis of DU145 cells treated with 17. Cells were treated with either
DMSO or 17 and the DNA content was measured by propidium iodide staining. To determine
the distribution of cells in various phases of cell cycle was taken as control. The percentage
of cells increased in G0/G1 phase up on exposure to 17 suggesting its role in G0/G1 arrest.
26

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Figure 6: Effect of 17 on activation of caspases inducing apoptosis in DU145 cells.
Treatment of DU145 cells with different concentration of 17 for 48 h induced activation of
caspases-3 (A) and caspase-9 (B) significantly in concentration dependent manner.
Doxorubicin was used as control for activation of both caspases. All experiments were
carried out in triplicates and mean values are presented here.
RHODAMINE
DMSO
FITC
MERGE
8.5μM
20μM
Figure 7: Effect of 17 on mitochondrial membrane potential inducing activation of caspase 9
was determined by staining the cells exposed to 17 with JC-1 dye. Cell treated with DMSO
show red fluorescence as the JC-1 dye is localized and aggregated in the mitochondrial
membrane. In cells exposed to 17, green fluorescence is increased where as red aggregated
fluorescence is decreased indicating depolarization of mitochondrial membrane.
27